Nerve growth factor (NGF) was first described by Buker (Anat. Rec. 102:369-389, 1948) and Levi-Montalcini (J. Exp. Zool. 116:321-362, 1951) as an activity secreted by a mouse sarcoma tumor implanted in a chick embryo. Both sensory ganglion and sympathetic ganglion neurons grew neurites into the sarcoma, which also supported the growth of peripheral neurons in culture. The factor, purified to homogeneity from mouse submandibular glands in 1956 by Levi-Montalcini and Cohen (Proc. Natl. Acad. Sci. U.S.A. 42:571, 1956) consists of a complex (referred to as 7S NGF, from its sedimentation coefficient) comprised of three different subunits. NGF's neurotrophic activity resides entirely within the .beta.-subunit (hereafter referred to as NGF), a dimer consisting of two equivalent monomers of approximately 13,000 dalton molecular weight.
A role for NGF as a neurotrophic factor in the peripheral nervous system (PNS) was rapidly established through both in vitro and in vivo experiments (Levi-Montalcini and Angeletti, Physiol. Rev. 48:534-569, 1968; Johnson, et al, Science 210:916-918, 1980). These studies demonstrated that sympathetic neurons of the PNS have an absolute requirement for NGF for survival throughout life, while many sensory neurons require NGF during certain periods of development. NGF is synthesized in the periphery by the nonneuronal target tissues innervated by the NGF-dependent neurons. Upon binding of NGF to its receptor, the NGF-receptor complex is internalized by the neuron and retrogradely transported back to the neuron cell body. NGF's intracellular mechanism of action is not yet fully elucidated.
It was not until 1983 that NGF was detected in the central nervous system (CNS) (Ayer-LeLievre, et al, Medical Biology 61:296-304, 1983). This discovery was preceded by the demonstration that the cholinergic neurons of the basal forebrain are responsive to NGF (Schwab, et al, Brain Res 168:473-483, 1979). These neurons possess NGF receptors which are indistinguishable from NGF receptors in the periphery. As in the PNS, NGF is synthesized by the target regions of the sensitive neurons, the hippocampus and the neocortex. NGF secreted by these target regions binds to its receptor and is internalized and transported back to the cholinergic cell bodies of the basal forebrain. The sensitivity of these neurons is especially interesting since these neurons are consistently depleted in Alzheimer's Disease (AD). It is possible, therefore, that an agent which enhances nerve growth factor's activity may be useful in treatment of CNS degenerative diseases like AD as well as peripheral neuropathies or other PNS degenerative disorders.
The term fibroblast growth factor may now be used to refer to any one of a family of peptide growth factors, but commonly refers to the best characterized members of the family, basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) (Klagsbrun, Prog. Growth Factor Res. 1:207-235). Both bFGF and aFGF are 154 amino acid peptides of approximately 18,000 molecular weight. In addition, higher molecular weight forms of bFGF, but not aFGF, are found in some tissues. bFGF is found in most tissues, while aFGF is located primarily in the brain. Neither bFGF nor aFGF contain signal peptide sequences, suggesting that these peptides are not secreted but are likely cell-associated and/or extracellular matrix proteins. Both peptides bind to heparin, a property that has been exploited for purification of FGFs. Both low and high affinity binding sites for the FGFs have been identified. The low affinity sites, found on cell surfaces and in extracellular matrix, are likely heparin-like molecules that serve to concentrate FGF (Moscatelli, J. Cell Physiol. 131:123-130, 1987). High affinity receptors have been identified in many cell and tissue types, including nervous tissue, and some studies suggest that both bFGF and aFGF and possibly other members of the FGF family bind to the same receptor (Neufeld and Gospodarowicz, J. Cell Physiol. 136:537-542, 1988; Basilico, et al, J. Cell Biochem. Suppl. 13b:78, 1989). Receptors for aFGF and bFGF have now been cloned from several species (Ruta, et al, Proc. Natl. Acad. Sci. U.S.A. 86:8722-8726, 1989; Lee, et al, Science 245:57-60, 1989; Reid, et al, Proc. Natl. Acad. Sci. U.S.A. 87:1596-1600, 1990). The receptor sequences contain putative tyrosine kinase domains and both aFGF and bFGF induce the phosphorylation of both high and low molecular weight proteins in intact cells (Friesel, et al, Mol. Cell Biol. 9:1857-1865, 1989; Coughlin, et al. J. Biol. Chem. 263:988-993, 1988), suggesting that this kinase activity represents the major signal transduction pathway for the FGFs.
Both bFGF and aFGF have been implicated in differentiation and development in many tissues. In addition, the angiogenic properties of both peptides suggest that they may play a role in wound healing. Of particular interest is an increasing body of data suggesting that the FGFs function to promote neuronal differentiation and survival. Both bFGF and aFGF have been shown to induce neurite outgrowth in PC12 cells (Togari, et al, J. Neurosci. 5:307-316, 1985) and neurons (Lipton, et al, Proc. Natl. Acad. Sci. U.S.A. 85:2388-2392, 1988). In addition, bFGF has been shown to imitate nerve growth factor's ability to rescue both the cholinergic neurons of the basal forebrain and retinal ganglion cells following surgical transection of their project (Anderson, et al, Nature 332:360-361, 1988; Sievers, et al, Neurosci Letters 76:157-172, 1987). Whether bFGF is acting directly on the neurons or indirectly via interactions with glia is as yet unknown. Regardless, these studies suggest that modulation of fibroblast growth factor activity may be beneficial in neurodegenerative diseases.
Both 5-bromo-4-(4-morpholinyl)-2-pyrimidinamine and 4-morpholino-2-piperazinothieno[3,2-d]pyrimidine and pharmaceutically acceptable salts thereof enhance the effects of nerve growth factor and 4-morpholino-2-piperazinothieno[3,2-d]pyrimidine and pharmaceutically acceptable salts thereof enhance the effects of fibroblast growth factor.